Photoresists processable under biocompatible conditions for multi-biomolecule patterning

ABSTRACT

Novel photoresist materials, which can be photolithographically processed in biocompatible conditions are presented in this invention. Suitable lithographic scheme for the use of these and analogous resists for biomolecule layer patterning on solid substrates are also described. The processes described enable micropatterning of more than two different proteins on solid substrates without denaturation of the proteins. The preferred resist materials are based on (meth)acrylate copolymers that contain at least one acid cleavable ester group and at least one hydrophilic group such as an alcoholic or a carboxylic group.

RELATED APPLICATIONS

This application is a National Stage entry of International ApplicationNumber PCT/GR02/00033, filed May 30, 2002, which claims priority toGreece Application 20010100271, filed May 31, 2001.

STATE OF THE ART

The photosensitive materials described in this invention are applied inthe microfabrication of bioanalytical devices where biomolecules areused for molecular recognition. These types of devices are referredusually as biosensors.

Based on the molecular recognition function, the biosensors are devidedin: a) biocatalytic sensors, when an enzyme recognizes an analytegenerating products by catalytic reaction and b) bioaffinity sensors,which are further devided in immunosensors and DNA sensors, when anantibody recognizes an antigen-analyte and a specific part of DNA chainrecognizes the complementary chain part as analyte, respectively. Due totheir signal trunsduction function, the biosensors are separated inelectrochemical semiconductor, piezoelectric, and optical sensors (1-2).

The patterning of many biomolecules on the same solid surface is veryimportant in bioanalysis, because it permits the simultaneous analysisof a large number of bioanalytes precisely and with low cost; it alsofacilitates the processing and the transduction of the recognitionsignal. To achieve this goal, most of the research effort is focused onthe thin film technology used in microelectronic device fabrication.However, a serious problem emerged from the transfer of this technologyto bioanalytic device fabrication is the non-compatibility ofphotolithographic processing with the presence of biomolecules. Anotherserious problem for pattering e.g. proteins on solid surface is not thechemistry itself required for protein immobilization, but the wholestrategy to prevent the adsorption of protein onto unwanted regions. Inthe following, the main methodologies that have been proposed to addressthese problems are presented.

Initially the biomolecules were used in photolithography to make surfacepatterning more convenient. Thus a denatured biomolecule layer wasformed between a photoresist film and silicon dioxide layer in order tomake easier the patterning of the silicon dioxide underlayer (3). Alsoan enzyme was dispersed in polymeric film for patterning metal byreactions depending on this enzyme (4).

The use of biomolecules in bioanalytic device fabrication was referredlater. In this context it was attempted to pattern enzyme membranephotolithographically by dispersing enzyme in photopolymer solution (5,6). The organic solvent developers, which are known to denaturesignificantly most of the biomolecules, in combination with theapplication of this method only in membranes limited this method.

A positive tone photoresist was used for direct patterning ofbiomolecules on substrates. During this procedure selective areas of theresist film were exposed to light and removed, then biomolecules wereintroduced to cover these regions and finally the unexposed film wasremoved with acetone (7). The use of acetone for the removal of theremaining film after biomolecule deposition limited decisevely theapplication of this “lift-off” technique.

Also, positive photoresists were used to pattern indirectly biomoleculeson substrates. According to this approach the photoresist film wasphotolithographically patterned, an alkylsilane covered both thesubstrate (exposed resist areas) and unexposed resist areas, theremaining film was stripped, an aminosilane was deposited on the regionsnot covered by alkylsilane and finally proteins or cells were adsorbedon the aminosilane stripes (8, 9). The whole strategy was veryeffective, but allowed patterning of only one type of biomolecules onsubstrate.

Another approach was the use of photoactivatable polymers for patterningbiomolecular assemblies (10-12). More particularly, patterned networkpolymers were formed upon substrates and subsequently biomolecules suchas antibodies or nucleic acid chains were bound on these networks. Thus,these polymers were used as photochemically activated linkers forcovalent binding of biomolecules. This strategy was not general and wasstrictly related to the kind of biomolecules (usually antibodies) to bebound, while it remains questionable if the multi-biomolecule patterningrequest can be fulfilled.

A different approach was the use of e-beam or deep UV photoresists fornegative and positive tone protein patterning (13). Thus, proteins werepatterned on the copolymer surface via e-beam or deep UV lithographywith two different mechanisms: the chemisorption- andphysisorption-controlled mechanism, which gave positive and negativeprotein images, correspondingly. The remaining photoresist after theprocedure and the obvious inability of patterning more than one proteinson the same substrate limited the application of this method. Otherpropositions have been reported using e-beam lithography withphotoresist in biodevice fabrication, but with potential patterning of asingle kind of biomolecules, due to high temperature thermal treatmentand organic solvent development preceded the biomolecule deposition(14).

Furthermore, several methods of photochemical surface modification forbiomolecule patterning without using photoresists have been reported.Characteristic category is the photochemical modification oforganosilane self-assembled monolayers (SAMs) for the formation ofcoplanar molecular assemblies, which are susceptible to selectivebiomolecule immobilization in micro scale (15-19). A similar approach isthe photochemical modification of polymer surface using cerium(IV)ammonium nitrate or psoralens for covalent immobilization ofbiomolecules (20, 21). A slightly different, but more complicated methodis the intercalative binding of psoralen moiety into DNA double strands,the light-induced DNA strands crosslinking through this moiety and thencopolymerization of the resulting DNA bearing vinyl groups with acomonomer introduced (22).

The use of photoisomerizable antigen monolayers for patterningantibodies on surfaces (23) and the light-induced protein immobilizationon avidin-photobiotin layer (24) are very interesting strategies, thoughnot general. An exceptional methodology for oligonucleotide arraysformation was the light-directed, spatially localized peptide synthesison solid phase (25). Concurrently the use of high-resolution imagingphotoresist in combination with the light-directed oligonucleotidesynthesis was resulted in significant increase of oligonucleotide arraysdensity (26).

The laser induced plasma vaporization and ionization technique wasproposed for electric field assisted deposition of proteins (27), butthe ionization of proteins is itself a limitation factor for the method.Also the protein patterning on gold-coated glass substrate through laserlithography was reported (28), while it is unknown if the biomoleculefunctionality was not affected by the extremely hot, ablated, goldregion.

A different approach for protein micropatterning was recently reportedby attaching proteins through chemical reactions within elastomericmicrofluidic networks (29). Also the micropatterning of differentproteins was achieved using a high-precision robot for the delivery ofnanoliter protein solution volumes onto chemically modified glass slidesfollowed by covalent binding of proteins on these spots (30).

Short Presentation of the Invention

A different approach of the above mentioned is presented in thisinvention: a chemically amplified photoresist based on the copolymerconsisted of the following monomers: 2-hydroxyethyl-methacrylate,isobornyl-methacrylate, t-butyl-methacrylate and acrylic acid (FIG. 1)is photolithographically processed under biocompatible conditions formicropatterning more than one proteins on solid substrate. The wholestrategy is a version of the “lift-off” lithography used inmicroelectronics, but fulfills the severe biocompatible requirementssuccessfully, due to the chemically amplified photoresist based on theinvented copolymer. The current methodology (the lithographic processand the photoresist) is a continuation of our research effort previouslyreported, in which a similar photolithographic lift-off technique wasfollowed, using, however, a different chemically amplified photoresistbased on the homopolymer t-butyl-acrylate (31, 32).

It is an object of this invention to micropattern one or more proteinson a substrate.

It is also an object of this invention to use the photolithographic“lift-off” technique in biocompatible conditions to micropattern one ormore proteins on a substrate.

It is still an object of this invention to make a copolymer basedphotoresist, which can be processed with the biocompatiblephotolithographic “lift-off” technique to micropattern one or moreproteins on a substrate.

It is also another object of this invention to make a copolymer basedphotoresist, which can be processed with the biocompatiblephotolithographic “lift-off” technique to micropattern one or moreproteins having resolution on the order of 1 to 100 microns onaminosilane-treated silicon surface.

It is still another object of this invention to make a copolymer-basedphotoresist, which can be processed with the biocompatiblephotolithographic “lift-off” technique to define one or more protein“bands” (parallel zones of proteins) on the internal surface ofpolystyrene or poly(methyl-pentene) capillary tube.

The above objects—except of the last object—are accomplished by thegeneral lithographic scheme that describes patterning of two proteins onsilicon surface treated with 3-aminopropyl-triethoxy-silane (APTES) inthe following steps (FIG. 2):

-   -   (1) Coating of the photoresist on the APTES-treated silicon        surface and subsequent thermal treatment of the film;    -   (2) Exposure of selected photoresist areas at specific        wavelength radiation;    -   (3) Dissolution of the previously exposed photoresist areas with        dilute aqueous base;    -   (4) Deposition of the active protein, which is adsorbed on the        substrate areas uncovered with the photolithographic steps (2)        and (3), and possibly on the unexposed film areas;    -   (5) Exposure at the same wavelength radiation of the remaining        photoresist film;    -   (6) Dissolution of the previously exposed remaining photoresist        by dilute aqueous base;    -   (7) Deposition of the inert protein, which is adsorbed on the        substrate areas uncovered with the lithographic steps (5) and        (6), and on the free binding sites of the areas where the active        protein was adsorbed.

Thus, two different proteins are patterned on the APTES-treated siliconsurface: the active protein is patterned on the substrate areas wherethe initially exposed photoresist regions were located, and the inertprotein is patterned on the other surface areas. (The inert protein is aprotein that does not interact with the fluorescent-labeled antibody,which is added to react with the patterned active protein after thewhole lithographic process for the visualization of the proteinmicrostructure). The steps that follow the deposition of the activeprotein molecules do not affect their immunoreactivity and are availablefor subsequent binding with their antibody molecules.

A slight modification of the previous lithographic scheme, which wouldpermit micropatterning of three different proteins on APTES-treatedsilicon surface and accomplish the same objects, is the processcomprising the following steps (FIG. 3):

-   -   (1) Coating of the photoresist on the APTES-treated silicon        surface and thermal treatment of the film;    -   (2) Exposure of selected photoresist areas at specific        wavelength radiation;    -   (3) Dissolution of the exposed areas with dilute aqueous base;    -   (4) Deposition of the first active protein, which is adsorbed on        the substrate areas uncovered with the photolithographic        steps (2) and (3), and possibly on the unexposed film areas;    -   (5) Deposition of the inert protein which is adsorbed on the        free binding sites of the substrate, and possibly on the        unexposed film areas;    -   (6) Exposure of other film areas at the same wavelength        radiation;    -   (7) Dissolution of the previously exposed film areas by dilute        aqueous base;    -   (8) Deposition of the second active protein, which is adsorbed        on the substrate areas uncovered with the photolithographic        steps (6) and (7), and possibly on the unexposed film areas;    -   (9) Exposure at the same wavelength radiation of the remaining        photoresist film;    -   (10) Dissolution of the previously exposed remaining film by        dilute aqueous base;    -   (11) Deposition of the inert protein, which is adsorbed on the        substrate areas uncovered with the lithographic steps (9) and        (10), and also on the free binding sites of the areas where the        two active proteins were adsorbed.

Thus, three different proteins are patterned on the APTES-treatedsilicon surface: the two active proteins are adsorbed on the substrateareas where the twice-selected exposed regions were located, and theinert protein is adsorbed on the other surface areas. Also the stepsthat follow the deposition of the two active protein molecules do notaffect their binding functionality. Modifying slightly the previouslithographic scheme four, five, etc. different proteins can be patternedon silicon surface without affecting their immunoreactivity.

The lithographic scheme that accomplishes the last object, thus,allowing the definition of protein “bands” on a plastic capillary innersurface is: the first of the two preceeding schemes if “bands” of twodifferent proteins have to be defined (FIG. 4), the second of the twopreceeding schemes if three proteins have to be defined, and so on. Thedifference now is the way that the process steps are carried out (e.g.introduction of the photoresist, the aqueous base or the proteinsolution with a syringe, the action of the capillary walls as radiationcut-off filter, etc).

BRIEF DESCRIPTION OF THE INVENTION FIGURES

FIG. 1. The chemical structure of the (meth)acrylate copolymer invented,which allow the photoresist to be photolithographically processed inbiocompatible conditions.

FIG. 2. The lithographic scheme for patterning two different proteins onAPTES-treated silicon surface with the biocompatible photolithographicprocessing (lift-off) of the (meth)acrylate photoresist.

FIG. 3. The lithographic scheme for patterning three different proteinson APTES-treated silicon surface with the biocompatiblephotolithographic processing (lift-off) of the (meth)acrylatephotoresist.

FIG. 4. The lithographic scheme for the definition of two protein“bands” on capillary inner surface with the biocompatiblephotolithographic processing (lift-off) of the (meth)acrylatephotoresist.

FIG. 5. Microstructures of 3.75 μm lines/spaces of two differentproteins: rabbit-IgG (green lines—active protein) and bovine serumalbumin (black lines—inert protein) obtained by the biocompatiblephotolithographic processing of the (meth)acrylate photoresist onAPTES-treated silicon surface.

FIG. 6. Microstructures of 22.5 μm lines/spaces of three differentproteins: mouse-IgG green lines—first active protein), biotinylatedbovine serum albumin (red lines—second active protein) and bovine serumalbumin (black lines—inert protein) resulted by the biocompatiblephotolithographic processing of the (meth)acrylate photoresist ontoAPTES-treated silicon surface.

FIG. 7 Two rabbit-IgG (active protein) “bands” defined among bovineserum albumin (inert protein) areas on poly(methyl-pentene) capillaryinner surface by the biocompatible photolithographic processing of the(meth)acrylate photoresist. The two protein “bands” are visualized byintroduction of anti-rabbit-FITC conjugate and subsequent fluorescencescanning of the whole capillary showing two distinct signals.

DETAILED DESCRIPTION OF THE INVENTION

In the first photolithographic scheme (FIG. 2) two proteins can bepatterned on silicon surface. Initially the silicon wafer surface istreated with 3-aminopropyl-triethoxy-silane (APTES) in order to becomehydrophobic for the subsequent physisorption of proteins. Then, thephotoresist solution is used for the creation of the photoresist filmonto the treated silicon surface. The photoresist solution constists oftwo compounds in an appropriate solvent: one component is the copolymerinvented and the other is a triphenyl or triaryl sulfonium salt acted asphotosensitiser. This solution is casted onto the treated silicon wafersurface and then the wafer is spinned in order to form a homogenicthickness film. Thermal treatment is followed for the vaporization ofthe solvent remained in the polymer matrix and the rearrangement of thepolymer chains. Although thermal treatment is not appropriate, usuallyit takes place for the formation of a high quality polymer film.

In the second step defined areas of the polymer film are exposed toradiation via a two-dimension photolithographic mask: a mask with atransparent pattern on its surface, which is transferred to the resistfilm. This mask must be kept in contact to the film (contact printing)to prevent phenomena of light diffraction, which would generate unwantedexposed areas. Also the radiation wavelength is selected in order tocontrol effectively the photo-induced reactions (the radiationwavelength must be in the photosensitiser absorption region). Thus,radiation band filters or cut-off filters are placed over the mask forthe selection of deep UV or near UV radiation region, respectively. Forthe chemically amplified resist as the current one, a thermal treatmentstep usually takes place after the exposure. But this photoresist, dueto the (meth)acrylate copolymer invented, does not need to bepostexposure treated and this is one of the main differences with ourpreviously introduced biocompatible photoresist (31, 32).

The third step is the removal of the previously exposed resist areasusing a very dilute aqueous base, which can be tolerated by theproteins. More particularly we had reported formerly (31,32) that theaqueous base solutions of 0.27 N tetramethyl ammonium hydroxide (TMAHconcentration (standard aqueous base developers used in semiconductortechnology) denature almost completely the proteins, while the 100 timesdiluted of this aqueous base solution (2.7×10³ N TMAH concentration)generates less than 10% decline of their immunoreactivity; this wasconsidered by us as a limit of protein denaturation and so this baseconcentration was the maximum value used. Actually the exposed areas ofthe current (meth)acrylate based photoresist can be dissolved in evenmore diluted aqueous base in comparison with our previous biocompatiblephotoresist (31, 32).

In the fourth step the patterned resist film is covered with the activeprotein solution for the adsorption of this protein on the substrate.Actually the protein is physisorbed not only on the substrate areas,where the photoresist areas were previously exposed and removed, butalso onto the remained unexposed film regions. The last one must not beconsidered as an inefficiency of the process, because the unexposedresist areas along with the protein monolayer adsorbed on them will beexposed and removed in the following steps. In this process the proteinsare adsorbed on the hydrophobic substrate, but it would be the same ifthe proteins were covalently bound on the surface without any changes inthe lithographic process. In addition it would cause no changes to theprocess sequence and conditions if other biomolecules were used insteadof proteins, such as enzymes, nucleic acid chain parts, etc.

The fifth step is consisted in the “flood” exposure of both the remainedphotoresist film and substrate areas where the proteins have beenadsorbed. The “flood” exposure is carried out without using a mask,because the whole film must be exposed, and not specific regions. Theradiation wavelength is the same with that in the previous exposure andis selected again using band filters or cut-off filters. It is necessaryfor this step that the radiation used must not affect the adsorbedproteins, because in the “flood” exposure they are irradiated togetherwith the remained photoresist. Thus, radiation in near UV or visible ispreferred with nearly no limitation in exposure time, while exposureduration in deep UV region must be kept relatively small. In additionthe duration of the second exposure is usually increased in comparisonwith the first one, because a small part of the photoresist surface isdeactivated in a way by the preceded covering of the film with theprotein solution; a surface dissolution of the film's photosensitiser isa possible explanation to this phenomenon. As with the second step nothermal treatment is needed after the photoresist exposure, againbecause of the incomparable dissolutive convenience of the exposed areasof our photoresist.

In the sixth step the dissolution of the previously exposed resist areasis taken place. The TMAH concentration of the aqueous base solution thatis used in this step may be slightly increased in comparison with thefirst dissolution made (third step), but it remains sufficiently belowthe base concentration level that is not tolerated by the proteins. Atthe end of this step a pattern of protein molecules is created on thesubstrate regions where the initially exposed resist areas (in thesecond step) were located.

In the seventh step the surface is covered by an inert protein solution.Therefore the inert protein molecules are adsorbed physically onto allfree surface binding sites: not only on the binding sites of thesubstrate regions uncovered with lithography in steps (5) and (6), butalso on the free binding sites that were not covered by the activeprotein molecules. The reason is to prevent non specific binding of theanalyte: the analyte (antibody) must be bound only onto the activeprotein (antigen) patterned and not adsorbed on the surface, in order toconfer the concentration of the analyte (use of the device inimmunoassay format). At the end of this step and of the whole processtwo proteins are patterned on the APTES-treated silicon surface: anactive protein that can be recognized by its antibody and an inertprotein that does not interact with this antibody. This pattern can bevisualized by the covering of the surface with the appropriate solutioncontaining the antibody labeled with a fluorescence substance andconsequently identification of the immunocomplex in an epifluorescencemicroscope (Example 1).

The process that allows the patterning of three proteins is the samewith that of patterning two proteins in relation to the general idea:the biocompatible photolithography of the (meth)acrylate basedphotoresist. The processing conditions of each step may have beenaltered and even the sequence of the steps, but this does not reduce thebiocompatibility and the effectiveness of the method. About thedifferentiation of the process steps sequence, it concerns only the“blocking” steps that is the steps whose purpose is to introducemolecules of an inert protein in order to occupy any uncovered substratesites. This step is taken place once at the end of the process, when twoproteins have to be patterned. Now for the process of patterning threeproteins the “blocking” step is taken place twice, not necessarily inthe same conditions: once immediately after the deposition of the firstactive protein and then again at the end of the process. Similarly iffour proteins have to be patterned the “blocking” step must take placethree times: firstly immediately after the deposition of the firstactive protein, secondly after the deposition of the second activeprotein and then again at the end of the process.

About the change of the process conditions, if the number of proteinsthat have to be patterned is increased this alteration is necessary forthe effective removal of the resist film. The adsorption of the proteinsonto the substrate is achieved, when the patterned photoresist isimmersed in slightly base solutions of proteins for a considerable time.Although these solutions are more diluted basic solutions than thoseused after every film exposure, they affect the resist filmsignificantly, because they cover it for a long time (may be more thanan hour is necessary). This causes deactivation of the polymer filmsurface layer and subsequent decrease in photoresist sensitivity,because of possible dissolution of the photoacid generator there, as itwas previously mentioned. Also the unwanted slightly exposed photoresistareas are more deactivated by the protein adsorption. That is why thelimitation of the light diffraction and consequently the exposure of theunwanted regions is an essential matter to the current photolithographicapproach. The radiation absorption by the protein monolayer physisorbedon the resist film must not contribute significantly to the polymer filmsurface deactivation, because the thickness of the protein monolayer isextremely small (in the order of 10 A) and consequently its absorptionis very small in comparison to the resist film.

To address the problem of surface deactivation of the polymer film thatis its sensitivity decline, less mild-though biocompatible-processconditions must be used for the removal of the film. Thus higherexposure duration is needed and/or slightly more concentrated basesolutions for the development of the film; usually it is preferred thefirst one and only if it is inevitable the second one. Consequently forthe three-protein patterning process the first exposure time might besmaller than the second one and the second one smaller than the thirdone. Similarly the first aqueous developer solution might have smallerbase (TMAH) concentration than the second one, the second one smallerthan the third one and the third one less to the one tolerated by theproteins (only 10% of immunoreactivity reduction is accepted; seeabove).

The three-protein pattern on the APTES-treated silicon substrate can bevisualized by two alternative ways: a) introduction of an appropriatesolution containing both antibodies of the two active proteins at theend of the photolithographic process or b) introduction of the twoantibodies separately: at first deposition of the first activeprotein-antibody after the corresponding “blocking” step (step 5) andthen deposition of the second active protein-antibody after the second“blocking” step (step 11). In both ways the antibodies are labeled witha different fluorescent substance so that each immunocomplex formed canbe identified in the epifluorescence microscope. Usually it is preferredthe first way of the above in order to avoid the effect of the antibodysolution on polymer film, but this depends on the antibodies too, e.g.if they can bind their corresponding antigens in the same pH solution,etc (Example 2).

When the biocompatible photolithographic process is applied for thepatterning of e.g. two proteins onto the plastic capillary internalsurface, extra limitations have been arisen, except the biocompatibleone. The solvent of the copolymer-based photoresist must not influencethe plastic material of the capillary tube, which is usuallypolystyrene, poly(methyl pentene), poly(methyl methacrylate), etc. Alsothe capillary walls act as cut-off filters and allow passing onlyradiation in near UV or visible region; consequently the photoresistfilm should be sensitive to this radiation region. The last requirementposed by the cylindrical geometry of the substrate forces us to use theappropriate photosensitisers, a problem that is not faced to the siliconsubstrate where deep UV exposure could be used, but in low doses inorder not to denature the patterned proteins.

Moreover practical problems concerning the application itself of thephotolithographic process are efficiently overcome. Thus the photoresistsolution is introduced in the capillary using a syringe, it is left inthe horizontal position for 1-2 min and then it is extracted by turningthe tube in the perpendicular position. The photoresist film formed canbe thermally treated by introduction of the capillary into aluminumplate holes of similar size with the capillary. After that, specificareas of the capillary external surface are exposed and the other areasare covered with a non-transparent tape; the use of radiation filters isnot necessary, since the capillary walls act as cut-off filters. Alsofor the homogeneous exposure of the photoresist film the capillary isturned by 60° around its axis at the end of each irradiation andconsequently the tube is turned five times in order to achieveirradiation of the whole film. Then the dissolution of the irradiatedresist areas is taken place by introducing the dilute aqueous basesolution with a syringe repeatedly (at least twice from both capillaryends in order to ensure that fresh developer solution is continuouslyadded). In a following step the protein solution is added, incubated forthe necessary period, and then rinsed. Afterwards, the whole resist filmis irradiated with the preceding homogeneous way (turning the capillaryby 60° after each exposure). Then the dilute aqueous base is introducedinto the capillary by a syringe (see above). Finally, the inert proteinsolution is introduced into the tube and incubated for a certain periodof time. For the visualization of the two protein locations onto thecapillary inner surface the specific antibody labeled with a fluorescentsubstance is introduced into the capillary and subsequent fluorescencescanning of the tube is taken place by an optical set-up constructed inour laboratory. Thus the locations containing the immunocomplex formedare indicated by different signals (Example 3).

The current photoresist is based on the copolymer:2-hydroxy-ethyl-methacrylate, isobornyl-methacrylate,t-butyl-methacrylate and acrylic acid. The chemical behavior of theresist is the same with the previously introduced photoresist based onthe homopolymer t-butyl acrylate. The reaction mechanism is the chemicalamplification mechanism, in which the acid generated by thephotosensitizer during the exposure acts as a catalyst for thesubsequent dissociation of the ester groups of the copolymer componentsin a way that for every ester pendant group that is broken with the aidof acid a new acid is generated. From the dissociation of the estergroup methacrylic acid is generated, which is soluble by diluted aqueousbase solutions. The extension of the ester groups dissociation definesthe following dissolution of the exposed photoresist areas. Thedifference between this photoresist and our previous one (31, 32) isthat now the dissolution of the exposed photoresist areas can beachieved without thermal treatment of the polymer film. Thephotosensitizers used are mainly two: a) triphenyl sulfoniumhexafluoroantinonate for the irradiation at deep UV (254 nm) or b) a 50%w/w solution in propylene carbonate of a 1:1 mixture of twotriarylsulfonium hexafluoroantimonate salts (one havingdiphenylthioether as an aryl substituent and the other is a thiodimer oftriphenyl sulfonium hexafluoroantimonate salt), for exposure at near UV(λ>300 nm).

The whole photolithographic strategy invented (both the copolymer-basedphotoresist and the biocompatible photolithographic “lift-off”technique) is a unique method for patterning biomolecules on solidsurface. It is a general methodology independent of the biomoleculesthat have to be patterned: proteins, enzymes or oligonucleotides can besimilarly patterned. Furthermore it is independent of the substratematerial and geometry: it can be applied on silicon or polymericsubstrate, in planar or cylindrical surface. Moreover, whatever way thebiomolecules are bound onto the substrate—adsorbed or covalentlybound—it is equally functional. No expensive or complicated instrumentsare needed; just the photoresist and the usual irradiation sources usedin deep UV or near UV exposure. And finally it can pattern in microscale easily more than one biomolecules on the same substrate. Inrelation to our former invented photolithographic strategy (31) the maindifferences are summarized to the following: a) the photoresist is basednow on the copolymer synthesized by us consisted of2-hydroxyethyl-methacrylate, isobornyl-methacrylate,t-butyl-methacrylate and acrylic acid in a 30/40/20/10 weight ratio,while the previous one was based on the homopolymer t-butyl acrylate, b)the photolithographic process fulfills better the biocompatibilityrequirements since using the copolymer synthesized lower exposure timesare necessary, no thermal treatment is needed after the exposure andeven more diluted aqueous bases can be used for the removal of theexposed photoresist areas. To show the effectiveness of this methodologysome characteristic examples are presented. In examples 1 and 2microstructures of two and three different proteinsphotolithographically patterned on APTES-treated silicon substrate areshown, correspondingly. In example 3 two “bands” of proteinsphotolithographically defined on the capillary inner surface arepresented.

EXAMPLES Example 1

Initially the silicon wafer surface is treated with3-aminopropyl-triethoxysilane (APTES) solution in order to make ithydrophobic (amino groups are formed) and susceptible to physicalbinding by the proteins. Thus a clean silicon wafer is immersed in a“pyranha” solution, for 1 hr, in ambient temperature; this solution is a1:1 mixture of 31% v/v H₂O₂ aqueous solution and 97% v/v H₂SO₄ aqueoussolution. Then the wafer is washed very well with deionized water and itis immersed in a bath with continuously refreshing deionized water(until its special resistance take the value of 12 MΩ). The water isremoved from the wafer surface under a nitrogen stream. Afterwards, itis immersed in 2% v/v APTES aqueous solution for 20 min and is quicklyimmersed and taken out from a bath with fresh deionized water, and driedunder a nitrogen stream; the reason that the wafer surface is immersednow in deionized water is to rinse it from the aminosilane salts thatare not bound to the surface. Subsequently the wafer is thermallytreated at 120° C. for 20 min. Finally it is immersed in deionized waterin ultrasonic bath for 5 min, dried under a nitrogen stream andthermally treated at 95° C. for 5 min (in order to remove completely thewater from the surface).

The photoresist solution is prepared as following: A 10% w/w solution inethyl lactate, of our synthesized copolymer consisted of2-hydroxyethyl-methacrylate, isobornyl-methacrylate,t-butyl-methacrylate and acrylic acid in a 30/40/20/10 weight ratio, isformulated. The solution is stirred at least for 1 hr with parallel mildthermal treatment (50° C.). Then triphenylsulfonium hexafluoroantimonatesalt, provided by General Electric, is added as photosensitizer, so thatthe final concentration of the salt to be 10% w/w in solids. The finalsolution is stirred for ½ hr and after that filtered (with filters of0.2 μm pore size).

To pattern two proteins—an active and an inert protein—onto the treatedsilicon surface the first photolithographic process is followed (FIG.2). Thus the above photoresist solution is cast on the center of thetreated silicon wafer covering about the ¾ of its surface. Thelithographic resist is coated on the whole silicon surface by spinningthe wafer at 3000 rpm for 30 sec. The coated wafer is baked in an ovenat 70° C. for 5 min. Selected areas from the resist film are exposedwith an Oriel Hg—Xe 500 W (operated at 450 W) radiation source. Theselection of the resist areas that are going to be patterned is donewith a quartz mask, which is placed over the polymer film and in contactwith it (through vacuum) for preventing light diffraction. The selectionof the wavelength radiation 254 nm (deep UV) is made with a broadbandfilter (50 nm bandwidth at half maximum), which is placed over the mask.The exposure dose is 36 mJ/cm². Subsequently the exposed areas aredissolved by immersion of the photoresist film in a 1.35×10⁻³ N aqueoussolution of tetramethyl ammonium hydroxide (TMAH for 2 min, washed withdeionized water and dried under a nitrogen stream. Then the wafersurface is covered with a 20 mg/L rabbit IgG solution in 0.04 Mphosphate buffer, pH 6.5, for 30 min. After that the surface is washedwith deionized water and dried under a nitrogen stream. Subsequently thewhole surface is exposed (“flood” exposure) using the same exposure tooland filter (254 nm broadband filter); the exposure dose is now 109mJ/cm². The remained film areas are dissolved by immersion of thephotoresist in a 2.7×10⁻³N TMAH aqueous solution for 2 min, washed withdeionized water and dried under a nitrogen stream. Finally the wholesurface is covered with a 10 g/L bovine serum albumin solution in 0.1 MNaHCO₃ buffer, pH 8.5, for 1 hr and dried with nitrogen stream. At theend of the process two proteins are patterned onto the APTES-treatedsilicon surface: rabbit IgG (active protein) and bovine serum albumin(inert protein).

To visualize the created protein pattern the substrate is immersed in a20 mg/L goat anti-rabbit IgG-fluorescein isothiocyanate conjugatesolution in 0.15 M Tris-HCl buffer, pH 8.25, containing 1 g/L bovineserum albumin, 0.5 g/L bovine IgG, 1 M KCl and 0.2 g/Lethylmercury-thiosalicylic acid sodium salt, and incubated for 3 days,at 4° C. Then the substrate is taken out, washed with a 0.01 M Tris-HClbuffer, pH 8.25, containing 0.05% (v/v) Tween 20, and observed inepifluorescence microscope. Microstructures of 3.75 μM lines/spaces oftwo proteins: rabbit-IgG (green lines) and bovine serum albumin (blacklines) are obtained (FIG. 5).

Example 2

In the beginning the silicon wafer surface is treated with3-aminopropyl-triethoxysilane (APTES) as it is described in Example 1.After that the photoresist solution is prepared. A 10% w/w solution inethyl lactate, of our synthesized copolymer consisted of2-hydroxyethyl-methacrylate, isobornyl-methacrylate,t-butyl-methacrylate and acrylic acid in a 30/40/20/10 weight ratio, isformulated. The solution is stirred at least for 1 hr with parallel mildthermal treatment (−50° C.). Then a 50% w/w solution in propylenecarbonate of a 1:1 mixture of two triarylsulfonium hexafluoroantimonatesalts (one having diphenylthioether as an aryl substituent and the otheris a thiodimer of triphenyl sulfonium hexafluoroantimonate salt),provided by Union Carbide with the name UVI 6974, is added to thecopolymer solution, so that the final concentration of the two saltstotally to be 30% w/w in solids. The ultimate solution is stirred for 30min and then filtered (with filters of 0.2 μm pore size).

The second photolithographic process (FIG. 3) is followed, because threedifferent proteins (two active and one inert) are going to be patterned.Thus the photoresist film is coated on the treated silicon wafer byspinning it at 3000 rpm for 30 sec. The film is baked in an oven at 95°C. for 5 min. The film areas that have to be exposed are selected byplacing a quartz mask over the film and in contact (through vacuum) withit. Also the required radiation region for λ>300 nm (near UV) is done byusing a pyrex cut-off filter over the mask. The exposure tool used is aKarl Suss aligner and the irradiation time is 5 min. Then the exposedareas are developed by immersion of the wafer in a 1.35×10⁻³ N aqueoussolution of tetramethyl ammonium hydroxide (TMAH) for 2 min, washingwith deionized water and drying under a nitrogen stream. Afterwards, thesurface is covered with a 50 mg/L mouse IgG solution in 0.04 M phosphatebuffer, pH 6.5, for 30 min; it is washed with deionized water and driedunder a nitrogen stream. Then it is covered with a 10 g/L bovine serumalbumin solution in 0.04 M phosphate buffer, pH 6.5, for 55 min, washedwith deionized water and dried under a nitrogen stream. Areas of thepatterned film are selected for exposure by covering some of theunexposed areas and leaving others to irradiation, but always using thepyrex cut-off filter. The exposure lasts again 5 min. The exposed areasare removed by immersion of the wafer in a 2.7×10⁻³ N aqueous TMAHsolution for 1 min; the substrate is washed with deionized water anddried under a nitrogen stream. After that the surface is covered with a20 mg/L biotinylated-bovine serum albumin solution in 0.04 M phosphatebuffer, pH 6.5, for 45 min; it is washed with deionized water and driedunder a nitrogen stream. Accordingly the whole surface is exposed(“flood” exposure) for 10 min with the pyrex filter. It is immersed in a2.7×10¹³ N aqueous TMAH solution for 5 min, rinsed with deionized waterand dried under a nitrogen stream. Thus, all the remained photoresistareas are dissolved. Finally the silicon surface is covered with a 10g/L bovine serum albumin solution in 0.1 M NaHCO₃ buffer, pH 8.5, for 2hr, washed with deionized water and dried with nitrogen stream. At theend of the photolithographic process three proteins are patterned on theAPTES-treated silicon surface: mouse IgG (first active protein),biotinylated-bovine serum albumin (second active protein) and bovineserum albumin (inert protein).

The protein pattern is visualized by incubation of the wafer in asolution of 20 mg/L goat anti-mouse IgG-fluorescein isothiocyanateconjugate and 5 mg/L streptavidin-R-phycoerytirin conjugate in 0.15 MTris-HCl buffer, pH 8.25, containing 5 g/L bovine serum albumin, 0.5 g/Lbovine IgG, for 2 days, at 4° C. After that the substrate is taken out,washed with a 0.01 M Tris-HCl buffer, pH 8.25, containing 0.05% (v/v)Tween 20 and observed using epifluorescence microscope. Microstructuresof 22.5 μm lines/spaces of three different proteins: mouse-IgG (greenlines), biotinylated bovine serum albumin (red lines) and bovine serumalbumin (black lines) are obtained (FIG. 6).

Example 3

For the definition of protein “bands” onto the internal surface of aplastic capillary tube the third biocompatible photolithographic process(FIG. 4) is used. The characteristics of the capillary, which is used inimmunoanalysis are the following: 6 cm length, 1 mm internal diameterand 1 mm walls thickness; the material is poly(methyl pentene). Thephotoresist solution used is the same with the Example 2: a 10% (w/w)solution of our synthesized copolymer in ethyl lactate with 30% (w/w insolids) of the 1:1 mixture of the two triarylsulfoniumhexafluoantimonate salts named UVI 6974.

Thus, the photoresist solution is introduced by a syringe into thecapillary, is left horizontally for 2 min and then is extracted byturning the capillary perpendicularly. The photoresist film formed has amedium thickness of ˜3 μm; a gentle nitrogen stream is used to removethe solution that blocks both edges of the capillary, while its externalsurface is cleaned with 2-propanol. Then a Karl Suss aligner exposuretool is used to expose two locations of the external capillary surface.For the selection of the exposed regions, two “rings” of 0.5 cm lengtheach one (1 cm distant each other and at 2 cm distance of each one fromthe capillary edge) are not covered and the other regions of theexternal capillary surface are covered with a non-transparent tape.Thus, the capillary is exposed for 1 min, then turned 60° around itsaxis, exposed again for 1 min, turned 60°, and so on until the wholefilm is irradiated (the capillary is turned 6 times by 60° each time andexposed 1 min for each time, too). Afterwards, the opaque tape isremoved and the tube is baked at 60° C. for 5 min into aluminum plateholes of similar size with the capillary. Then a 2.7×10⁻³ N aqueoussolution of tetramethyl ammonium hydroxide (TMAH) is introduced in acontinuous way into the capillary: 4 times by 2 ml solution each time,twice from each edge and with 5 min total time of the development. Thewashing of the capillary with deionized water is followed with the samecontinuous way (4×2 ml for 5 min) and the tube is dried under a nitrogenstream. Then, a 20 mg/L rabbit IgG solution in 0.04 M phosphate buffer,pH 6.5, is introduced into the capillary and incubated for 30 min. Thecapillary is washed with deionized water as described above (4×2 ml for5 min) and dried with nitrogen stream. Subsequently the whole capillaryis exposed with the previous manner (6×1 min exposure dose with 60° turnof the capillary each time). After that it is baked at 60° C. for 5 minin the aluminum plate with the holes. Next a 2.7×10⁻³ N aqueous TMAHsolution is introduced into the capillary as described above (4×2 ml for5 min), it is washed with deionized water with the same manner and driedunder a nitrogen stream. Then a 10 g/L bovine serum albumin in 0.1 MNaHCO₃ buffer, pH 8.5, is incubated in the tube for 1 hr; subsequentlythe capillary is dried with nitrogen. At the end of the lithographicprocess two distinct “zones” of rabbit IgG (active protein) are formedand the other areas of capillary inert surface are covered with bovineserum albumin (inert protein).

For the visualization of the above “bands” the capillary is filled witha 5 mg/L goat anti-rabbit IgG-fluorescein isothiocyanate conjugatesolution in 0.15 M Tris-HCl buffer, pH 8.25, containing 1 g/L bovineserum albumin, 0.5 g/L bovine IgG, 1 M KCl and 0.2 g/Lethylmercury-thiosalicylic acid sodium salt, and incubated for 30 min,at 22° C. Then it is washed (4×1 ml) with a 0.01 M Tris-HCl buffer, pH8.25 containing 0.05% (v/v) Tween 20 and dried under a nitrogen stream.Afterwards, the capillary is filled with the same washing solution, andthe fluorolabeled immunocomplex formed on the internal capillary surfacewas determined by scanning the capillary perpendicularly with a specificoptical set-up constructed in our laboratory. Two distinct signals areobtained indicating two areas with the fluorolabeled immunocomplexformed.

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1. A kit of photolithographic processing reagents comprising: (1)photoresist comprising a copolymer synthesized from monomers, wherein atleast one of said monomers is 2-hydroxyethyl-methacrylate and wherein atleast one of said monomers contains an acid cleavable group and at leastone of said monomers contains a hydrophilic group selected from theclass of hydroxyls and carboxyls; and (2) dilute aqueous base developerof less than 0.01 N base concentration.
 2. Method of photolithographicprocessing using photoresist comprising a copolymer synthesized frommonomers, wherein at least one of said monomers contains an acidcleavable group and at least one of said monomers contains a hydrophilicgroup selected from the class of hydroxyls and carboxyls, comprising thesteps of exposure of selected photoresist areas, dissolution of saidphotoresist areas and deposition of biomolecules on the substrate,wherein the step of dissolution is accomplished using dilute aqueousbase developer that is less than 0.01 N base concentration and whereinexposure of selected photoresist areas and the subsequent dissolution ofsaid photoresist areas follows the deposition of biomolecules on thesubstrate without deactivating the deposited biomolecules.
 3. Methodaccording to claim 2, wherein the exposure of selected photoresist areasand the subsequent dissolution of said photoresist areas is repeatedmore than once without deactivating the deposited biomolecules. 4.Method of photolithographic processing using photoresist comprising acopolymer synthesized from monomers of 2-hydroxyethyl- methacrylate,isobornyl-methacrylate, t-butylmethacrylate, and acrylic acid, whereinthe method comprises the steps of exposure of selected photoresistareas, dissolution of said photoresist areas and deposition ofbiomolecules on the substrate.
 5. Photoresist in contact with adeveloper wherein said photoresist comprises a copolymer synthesizedfrom monomers, wherein at least one of said monomers is2-hydroxyethyl-methacrylate and wherein at least one of said monomerscontains an acid cleavable group and at least one of said monomerscontains a hydrophilic group selected from the class of hydroxyls andcarboxyls; and wherein said developer is dilute aqueous base developerof less than 0.01 N base concentration.
 6. Method of photolithographicprocessing comprising the steps of exposure of selected photoresistareas, dissolution of said photoresist areas and deposition ofbiomolecules on a substrate wherein the step of dissolution isaccomplished with the photoresist in contact with a developer, asrecited in claim
 5. 7. Method of photolithographic processing formicropatterning biomolecules that are proteins or nucleic acid chainparts on a substrate comprising the steps of exposure of selectedphotoresist areas, dissolution of said photoresist areas and depositionof biomolecules on the substrate wherein the step of dissolution isaccomplished with a photoresist in contact with a developer, as recitedin claim
 5. 8. The photoresist comprising a copolymer synthesized frommonomers of 2-hydroxyethyl-methacrylate, isobornyl-methacrylate,t-butylmethacrylate, and acrylic acid wherein the proportion of2-hydroxyethyl-methacrylate/isobornyl-ethacrylate/t-butylmethacrylate/acrylic acid is 30/40/20/10 by weight.